For several years it has been known that enkephalin biosynthesis occurs via the ribosomal process. However, until recently no larger polypeptides containing the enkephalin sequence have been isolated. We have identified in both the adrenal medulla and striatum a number of polypeptides containing enkephalin sequences that are released upon trypsin proteolysis. These range in size from 3.9 Kdal to 80 Kdal and contain from one to eight enkephalin sequences. In the adrenal medulla these polypeptides are present in the chromaffin granules and are released concomitantly with the catecholamines. From bovine adrenal chromaffin granules four of these polypeptides have been purified and partially characterized. The goals of this project are to purify the remaining enkephalin-containing polypeptides from bovine adrenal and to establish the pathway leading from the largest precursor to the enkephalins. In addition, the precursor polypeptides from sheep and rat adrenals and from striata will be isolated and compared to those from beef. This will provide assurance that the pathway defined in beef is a universal pathway. Using antibodies to the various polypeptides their levels in tissue and in the circulation can be measured. Using the micromethodology and HPLC technology we have developed, relatively small amounts of tissue are suffient for purification and characterization. Sequence homology between precursor polypeptides will be established initially by HPLC tryptic mapping with sequencing data used where necessary. Assays for enkephalin-containing polypeptides will be done using both radioimmunoassay and radioreceptor binding assay. Antibodies to the purified polypeptides will be raised in rabbit or possibly by monoclonal techniques. In addition to the importance of the enkephalins as neuromodulators, we have preliminary data suggesting that the smaller enkephalin-containing polypeptides (i.e. 3,500-8,000 daltons) have neurohormonal activities of their own and are not merely enkephalin precursors. If the polypeptides from different species are similar in sequence it will provide further evidence for neurohormonal roles for them. Studies could then be initiated to determine their physiological activities. The characterization of the biosynthetic pathway leading to the enkephalins and to these possible new neurohormones can provide a basis for identifying the roles these peptides play in neurological disorders.